Extended-spectrum Β-lactamase Genes Research Articles

Carbapenemase and ESBL genes with class 1 integron among fermenting and nonfermenting bacteria isolated from water sources from India.

The present study was aimed to detect the carbapenemase, extended-spectrum β-lactamase (ESBL), and intI1 gene of class 1 integron among fermenting (n=61) and nonfermenting (n=10) bacterial isolates recovered from water samples (n=128). Isolates were identified by 16S rRNA sequencing. These isolates showed reduced-susceptibility to third-generation cephalosporins and carbapenems. The isolates varied in number and size of plasmids (2kb to >20kb). Plasmid DNA screening showed 5·6, 7, 11·2 and 26·7% prevalence of blaKPC , blaNDM , blaSHV and blaTEM genes respectively. Diverse blaNDM (blaNDM-1 and blaNDM-4 ) and blaSHV subtypes (blaSHV-2 and blaSHV-11 ) were recorded, unlike the single allelic blaKPC (blaKPC-2 ) and blaTEM (blaTEM-1 ) gene. Of the total 27 bla-gene-producing bacterial isolates, seven isolates co-harboured the carbapenemase genes (blaNDM or blaKPC or the both) along with the ESBL genes (blaSHV or blaTEM ). The intI1 gene of class 1 integron was detected among 12 (44·4%) of ESBL- and/or carbapenemase-harbouring isolates. Gene transferability was seen among four of the 10 Enterobacteriaceae donors. Carbapenemases and ESBLs with class 1 integron among aquatic environmental isolates raise the serious issue of the biosecurity and health of the ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: Anthropologically affected and polluted environment harbours the resistance threats, where a diverse bacterial species maintain, develop and exchange genetic determinants that constitute a risk to human and ecological health. The antimicrobial resistance (AMR) in Enterobacteriaceae and non-Enterobacteriaceae bacteria caused the failure of the therapy of last resort (carbapenems) and thus lead to life-threatening infections affecting public health. Surveillance and monitoring of AMR could be important for epidemiological, diagnostic testing and control of pathogens. This is a point-prevalence study reporting the comparative occurrence and co-occurrence of carbapenemase and extended-spectrum β-lactamase genes among fermenting and nonfermenting bacteria isolated from the aquatic environment in India.

Complex Class 1 Integron in a Clinical Escherichia coli Strain From Vietnam Carrying Both mcr-1 and bla NDM-1.

The co-production of MCR and carbapenemase in Enterobacteriaceae has been previously reported. Here, we describe a clinical strain of Escherichia coli from Vietnam carrying both mcr-1 and blaNDM–1. Whole-genome sequencing showed that the genome of this strain consists of a 4,975,832-bp chromosome and four plasmids. The mcr-1 and blaNDM–1 genes are located on IncI2 and IncA/C2-type plasmids, respectively. Genetic analysis revealed the presence of a multidrug-resistant region with the structure of a novel complex class 1 integron including a class 1 integron region bearing two 5′ conserved segments and one 3′ conserved segment and two complete structures of ISCR1. The complex integron contains aminoglycoside resistance genes aadA2, aadB, strA, strB, and aphA6, quinolone resistance gene qnrA1, extended-spectrum β-lactamase gene blaOXA–4, and a Tn125-like transposon bearing blaNDM–1. In addition, the dfrA12-gcuF-aadA2-cmlA1-aadA1-qacH gene cassette array belonging to the sul3-type integron was also identified, but the region found downstream of the gene cassette array is the IS440-tet(M)-IS26 element instead of the sul3 gene. The results further support that Enterobacteriaceae isolates co-harboring mcr and blaNDM are widely being distributed. The structural characteristics of the complex integron reveal that ISCR1 elements play an important role in the mobilization of blaNDM–1 and the development of multidrug-resistant regions.

1436. Comparison of Ceftazidime–Avibactam, Ceftolozane–Tazobactam, Piperacillin–Tazobactam, and Meropenem Activities When Tested Against Gram-Negative Organisms Isolated From Complicated Urinary Tract Infections

BackgroundComplicated urinary tract infections (cUTIs) represent a major cause of healthcare-associated infection and a major source of gram-negative (GN) bacteremia. We evaluated the antimicrobial activities of recently approved β-lactamase inhibitor combinations and comparators against GN bacteria isolated from patients with cUTIs in the US hospitals in 2018.MethodsUnique patient isolates were consecutively collected from patients with cUTIs in 65 hospitals in 2018, and the GN organisms (n = 4,371) were susceptibility (S) tested by reference broth microdilution methods. Enterobacterales (ENT) with elevated cephalosporin MICs were screened for β-lactamase-encoding genes by whole-genome sequencing.ResultsThe most common GN organisms were E. coli (44.5%), K. pneumoniae (19.6%), P. mirabilis (6.7%), and P. aeruginosa (PSA; 5.3%). The most active agents against ENT were ceftazidime–avibactam (CAZ-AVI; 99.9%S), amikacin (AMK; 99.7%S), and meropenem (MEM; 99.4%S; table). Extended-spectrum β-lactamase (ESBL) genes were identified in 315 ENT (7.6%; excluding carbapenemase co-producers), including CTX-M-15 (63% of ESBL producers), other CTX-M types (25%), OXA-1/OXA-30 (39%), and SHV type (30%); approximately 50% of ESBL producers had ≥2 ESBL genes, mainly a CTX-M-type and an OXA-type (37% of isolates). The most active agents against ESBL producers were CAZ-AVI (100.0%S), AMK (99.7%S), and MEM (99.4%S); whereas ceftolozane–tazobactam (C-T) and piperacillin–tazobactam (PIP-TAZ) were active against 90.6% and 84.8% of ESBL producers, respectively. Only CAZ-AVI (87.0%S), colistin (COL; 87.0%S), and tigecycline (95.7%S) exhibited good activity against carbapenem-resistant ENT (CRE). Only 3 ENT isolates (0.07%) were CAZ-AVI resistant and all had a metallo-β-lactamase gene (2 VIM-1 and 1 NDM-1). CAZ-AVI (97.0%S) and C-T (99.1%S) were the most active β-lactams tested against PSA; other compounds with > 90%S for PSA were COL (99.6%), AMK (97.8%), tobramycin (93.5%), and CAZ (90.4%).ConclusionCAZ-AVI was highly active against a large collection of contemporary GN bacteria isolated from patients with cUTIs in US hospitals and provided greater coverage than the agents currently available in the US to treat cUTIs. Disclosures All authors: No reported disclosures.

623. Antimicrobial Resistance in Non-Typhoidal Salmonella from Retail Poultry Meat by Antibiotic Usage-related Production Claims—Pennsylvania, 2008–2017

BackgroundAntimicrobial-resistant (AMR) nontyphoidal Salmonella infections are a public health concern. Injudicious use of antimicrobials fuels emergence of resistance. The National Antimicrobial Resistance Monitoring System (NARMS) tracks AMR in Salmonella from humans, animals and foods. There is limited evidence regarding antimicrobial use in food animals and AMR bacteria in retail meat.MethodsWe reviewed antimicrobial susceptibility and whole-genome sequencing data from 320 Salmonella isolated from poultry meat in Pennsylvania as part of NARMS activities. Salmonella strains were isolated from 3,481 samples purchased from randomly selected retail outlets during 2008–2017. Antibiotic usage claims on meat packages were used to compare AMR Salmonella from conventional and antibiotic-free/organic (Abx-free) samples. Genetic mechanisms for AMR were investigated in a subset of isolates.ResultsThe prevalence of Salmonella in conventional poultry meat 10.2% (280/2,733) was significantly higher than the prevalence in poultry meat labeled as Abx-free (5.3%, 40/748; P < 0.0001). Salmonella from conventional poultry meat was more likely to be resistant to 3 or more drugs (55.0%, 154/280) compared with poultry meat labeled as Abx-free (27.5%, 11/40; P = 0.0011). Salmonella from conventional poultry exhibited significantly higher resistance to 4 drug classes including β-lactams (P = 0.006) (figure). One hundred isolates from conventional poultry meat and 8 isolates from antibiotic-free/organic samples harbored a gene conferring resistance to the β-lactam class; 24.3% (68/280) of isolates from conventional and 7.5% (3/40) of isolates from Abx-free samples (P = 0.0145) contained the extended-spectrum β-lactamase (ESBL) gene blaCMY-2.ConclusionMeat samples from conventionally-raised poultry were more likely to be contaminated with AMR Salmonella strains and have genes that reduce the effectiveness of antimicrobial drugs recommended for treatment of severe infections. Contamination of poultry with AMR Salmonella strains is concerning as is the presence of genes that decrease the power of critical antibiotics such as β-lactams. These findings highlight the importance of judicious use of antibiotics in food-producing animals. Disclosures All authors: No reported disclosures.

624. Molecular Characterization of Baseline Enterobacteriaceae and Pseudomonas aeruginosa from a Phase 3 Nosocomial Pneumonia (ASPECT-NP) Clinical Trial

BackgroundASPECT-NP, a phase 3, randomized, double-blind, multicenter trial, evaluated ceftolozane/tazobactam (C/T) 3 g q8h vs. meropenem 1 g q8h for 8–14 days in adults for treatment of ventilated nosocomial pneumonia. Baseline Gram-negative (GN) isolates from patients were tested for mechanisms of resistance.MethodsLower respiratory tract (LRT) isolates were sent to a central laboratory for organism identification and susceptibility. Of 664 total Enterobacteriaceae (ENT) and Pseudomonas aeruginosa (PsA) isolates, 351 (53%) were nonsusceptible to broad-spectrum cephalosporins and/or carbapenems and underwent whole-genome sequencing, quantitative RT–PCR, and western blot analysis. ENT isolates were tested for the presence of acquired β-lactamase genes and AmpC levels (selected species). PsA isolates were tested for acquired β-lactamase genes, AmpC (PDC) levels, efflux pump expression, and OprD loss.ResultsOf 262 ENT isolates, 114 (44%) were susceptible to C/T (MIC ≤2 µg/mL). An extended-spectrum β-lactamase (ESBL) gene was carried by 89 (78%) of the C/T-susceptible isolates. Of 148 C/T-nonsusceptible (C/T-NS) isolates, 87 (59%) were carbapenemase negative, and the majority 135 (91%) also carried an ESBL gene. The most common ESBL was blaCTX-M15 with blaOXA-1 and blaOXA-30. Klebsiella pneumoniae often displayed higher C/T MICs compared with other species carrying the same resistance genes. Among all C/T-NS isolates, 61 (41%) were carbapenemase positive, most commonly K. pneumoniae carrying blaOXA-48, blaNDM-1, and blaNDM-5. Of 89 PsA isolates, 58 (65%) were susceptible to C/T (MIC ≤4 µg/mL), despite elevated AmpC expression, efflux pumps, or loss of OprD; only 5 isolates had an acquired β-lactamase. Of the 31 C/T-NS PsA isolates, only 12 (39%) were carbapenemase positive and carried blaVIM or blaGES; isolates carrying blaGES had lower C/T MICs (8–32 µg/mL) compared with blaVIM (MIC > 128 µg/mL). PDC alleles were similar in isolates with high and low C/T MICs.ConclusionIn baseline GN LRT isolates from ASPECT-NP, the most common ESBL detected in ENT was blaCTX-M15; carbapenemases were uncommon. There was no correlation of ESBL phenotype to C/T susceptibility among ENT, nor of PDC allele to C/T susceptibility among PsA.Disclosures All authors: No reported disclosures.

First report of Burkholderia pseudomallei ST412 and ST734 clones harbouring blaOXA-57 but susceptible to imipenem in India.

Melioidosis caused by Burkholderia pseudomallei has become an important clinical threat, especially in Northern Australia and Southeast Asia. However, the genome information on this pathogen is limited. B. pseudomallei isolates identified from bloodstream infections from inpatients were subjected to whole-genome sequencing by IonTorrent PGM and MinION Oxford Nanopore sequencing technologies. Highly accurate complete genomes of two strains, VB3253 and VB2514, were obtained by a hybrid genome assembly method using both short and long DNA reads. Both isolates carried blaPenI and carbapenemase-encoding blaOXA-57 genes, although the isolates were susceptible to imipenem by E-test method with MIC 1 μg/mL. Multiple IS family transposases specific for all non-fermenting Gram-negative bacteria (NFGNBs)—especially IS3 and IS5, which facilitate mobilization of extended-spectrum β-lactamase (ESBL) and carbapenemase genes—were carried in these genomes. This further adds to the complexity of gene transmission. These IS families were identified only upon hybrid genome assembly and would otherwise be missed.

Occurrence of Virulence and Extended-Spectrum β-Lactamase Determinants in Vibrio spp. Isolated from Marketed Hard-Shelled Mussel (Mytilus coruscus).

This study investigated the occurrence of virulence and antimicrobial resistance properties in Vibrio spp. isolated from hard-shelled mussel (Mytilus coruscus) marketed in Korea. A total of 32 Vibrio spp. isolates including Vibrio diabolicus (n = 16), Vibrio alginolyticus (n = 13), Vibrio parahaemolyticus (n = 2), and Vibrio harveyi (n = 1) were identified by gyrB gene sequencing. Every isolate grouped with their respective reference sequence excluding V. diabolicus and V. alginolyticus that were intermixed in a neighbor-joining phylogenetic tree. Every Vibrio spp. showed DNase and gelatinase activities. Phospholipase, slime, caseinase, lipase, β-hemolysis, and α-hemolysis were positive in 31 (96.8%), 31 (96.8%), 29 (93.5%), 28 (87.5%), 17 (53.1%), and 9 (28.1%) isolates, respectively. The PCR amplification of virulence genes displayed that V. alginolyticus-specific (toxR, tlh, VAC, and VPI) genes were detected in 23 (71.9%), 18 (56.3%), 21 (65.6%), and 26 (81.3%) isolates, respectively. V. parahaemolyticus-specific (toxR and tlh) genes were harbored by 2 (6.3%) and 2 (6.3%) isolates, respectively. The other virulence genes including ctxAB and hupO genes were observed in 23 (71.9%) and 16 (50.0%) isolates, respectively. The antimicrobial resistance was prevalent for amoxicillin (100.0%), ampicillin (100.0%), and streptomycin (37.5%) in disk diffusion test. Multiple antimicrobial resistance index ranged from 0.09 to 0.22. The extended-spectrum β-lactamase (ESBL) genes, blaCTX, blaTEM, blaSHV, and blaOXA were detected in 28 (87.5%), 13 (40.6%), 7 (21.8%), and 1 (3.1%) isolates, respectively. Non-ESBLs such as streptomycin resistance (strA.B), kanamycin resistance (aphA-IAB), and tetracycline resistance (tetE) genes were found in 5 (15.6%), 3 (9.4%), and 1 (3.1%) isolates. Class 1 integron-related Integrase 1 (intI1) gene was found in 8 (25.0%) isolates. Our results reveal that the consumption of raw mussels may cause a potential public health risks owing to the virulent and antimicrobial-resistant Vibrio spp.

Study of Antibiotic Susceptibility Pattern and Frequency of Extended Spectrum β-lactamases Genes TEM and SHV in Urinary Tract Isolates of Escherichia coli and Klebsiella pneumoniae in Tabriz

Study of Antibiotic Susceptibility Pattern and Frequency of Extended Spectrum β-lactamases Genes TEM and SHV in Urinary Tract Isolates of Escherichia coli and Klebsiella pneumoniae in Tabriz

Genetic relatedness in carbapenem-resistant isolates from clinical specimens in Ghana using ERIC-PCR technique.

AimEnterobacterial repetitive intergenic consensus (ERIC) sequence analysis is a powerful tool for epidemiological analysis of bacterial species. This study aimed to determine the genetic relatedness or variability in carbapenem-resistant isolates by species using this technique.MethodsA total of 111 non-duplicated carbapenem-resistant (CR) Gram-negative bacilli isolates from a three-year collection period (2012–2014) were investigated by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC–PCR) in four selected hospital laboratories in Ghana. The isolates were also screened for carbapenemase and extended spectrum β-lactamase genes by PCR.ResultsA proportion of 23.4% (26/111) of the genomic DNA extracts were carriers of PCR-positive carbapenemase genes, including 14.4% blaNDM-1, 7.2% blaVIM-1 and 1.8% blaOXA-48. The highest prevalence of carbapenemase genes was from non-fermenters, Acinetobacter baumannii and Pseudomonas aeruginosa. For the ESBL genes tested, 96.4% (107/111) of the CR isolates co-harboured both TEM-1 and SHV-1 genes. The ERIC-PCR gel analysis exhibited 1 to 8 bands ranging from 50 to 800 bp. Band patterns of 93 complex dissimilarities were visually distinguished from the 111 CR isolates studied, while the remaining 18 showed band similarities in pairs.ConclusionOverall, ERIC-PCR fingerprints have shown a high level of diversity among the species of Gram-negative bacterial pathogens and specimen collection sites in this study. ERIC-PCR optimisation assays may serve as a suitable genotyping tool for the assessment of genetic diversity or close relatedness of isolates that are found in clinical settings.

Genome Analysis of Multidrug-Resistant Escherichia coli Isolated from Poultry in Nigeria.

Escherichia coli is one of the most common commensal bacteria of the gastrointestinal tract of humans and warm-blooded animals. Contaminated poultry can lead to disease outbreaks in consumers causing massive economic losses in the poultry industry. Additionally, commensal E. coli can harbor antibiotic resistance genes that can be transferred to other bacteria, including pathogens, in a colonized human host. In a previous study on antimicrobial resistance of E. coli from food animals from Nigeria, multidrug-resistant E. coli were detected. Three of those isolates were selected for further study using whole-genome sequencing due to the extensive drug resistance exhibited. All of the isolates carried the extended-spectrum β-lactamase (ESBL) genes, blaCTX-M15 and blaTEM-1, whereas one isolate harbored an additional ESBL, blaOXA-1. All of the tetracycline-resistant isolates carried tet(A). The genes aac3-IIa and aacA4, conferring resistance to aminoglycosides, were identified in an E. coli isolate resistant to gentamicin and tobramycin. In two E. coli isolates, dfrA14, qnrS1, and sulII, were detected conferring resistance to trimethoprim, fluoroquinolones, and sulfonamides, respectively. The third isolate carried dfrA17, no fluoroquinolone resistance gene, an additional sulI gene, and a chloramphenicol resistance gene, catB3. Mutations in candidate genes conferring resistance to fosfomycin and fluoroquinolones were also detected. Several efflux systems were detected in all the E. coli isolates and virulence-associated genes related to serum resistance, motility, and adhesion. E. coli and non-E. coli origin prophages were also identified in the isolates. The results underline the higher resolution power of whole-genome sequencing for investigation of antimicrobial resistance, virulence, and phage in E. coli.

Raw wastewater irrigation for urban agriculture in three African cities increases the abundance of transferable antibiotic resistance genes in soil, including those encoding extended spectrum β-lactamases (ESBLs)

A study was conducted to investigate the impact of raw wastewater use for irrigation on dissemination of bacterial resistance in urban agriculture in African cities. The pollution of agricultural fields by selected antibiotic residues was assessed. The structure and functions of the soil microbial communities, presence of antibiotic resistance genes of human clinical importance and Enterobacteriaceae plasmid replicons were analysed using high throughput metagenomic sequencing. In irrigated fields, the richness of Bacteroidetes and Firmicutes phyla increased by 65% and 15.7%, respectively; functions allocated to microbial communities' adaptation and development increased by 3%. Abundance of antibiotic resistance genes of medical interest was 27% greater in irrigated fields. Extended spectrum β-lactamase genes identified in irrigated fields included blaCARB-3, blaOXA-347, blaOXA-5 and blaRm3. The presence of ARGs encoding resistance to amphenicols, β-lactams, and tetracyclines were associated with the higher concentrations of ciprofloxacin, enrofloxacin and sulfamethoxazole in irrigated fields. Ten Enterobacteriaceae plasmid amplicon groups involved in the wide distribution of ARGs were identified in the fields. IncQ2, ColE, IncFIC, IncQ1, and IncFII were found in both farming systems; IncW and IncP1 in irrigated fields; and IncY, IncFIB and IncFIA in non-irrigated fields. In conclusion, raw wastewater irrigated soils in African cities could represent a vector for the spread of antibiotic resistance, thus threatening human and animal health. Consumers of products from these farms and farmers could be at risk of acquiring infections due to drug-resistant bacteria.

Genomic Analysis Of A KPC-2-Producing Klebsiella Pneumoniae ST11 Outbreak From A Teaching Hospital In Shandong Province, China.

PurposeKlebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bacteria causes nosocomial infections worldwide. However, KPC-producing K. pneumoniae outbreak has never been reported in Shandong Province, China. The purpose of our study was to elucidate the epidemiological and drug resistance mechanisms of KPC-producing K. pneumoniae strains collected from a large teaching hospital in Shandong during the outbreak. Moreover, we attempted to characterize the genetic environment and phylogenetic analysis of blaKPC-2 in outbreak isolates.MethodsWe monitored a 64-day outbreak of infection in a general hospital in Shandong Province, and the bacteria causing the infection were all ST11-type K. pneumoniae. The genotype correlation of KPC-producing K. pneumoniae isolates was assessed by whole-genome sequencing (WGS) phylogenetic analysis. Subsequent studies included antibiotic susceptibility testing, multilocus sequence typing (MLST) and S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blot hybridization.ResultsFrom February 1, 2018 to April 5, 2018, 14 KPC-producing K. pneumoniae isolates from different wards were collected. All 14 isolates were resistant to carbapenems and carried the extended-spectrum β-lactamase (ESBL) gene as well as fosA, and sul genes. Whole-genome analysis showed that all 14 the outbreak isolates were all ST11 type. The blaKPC-2 carrying plasmids were all belong to IncFIIK2 type, and the size ranged from 94 kb to 368 kb.ConclusionAs far as we know, this report first describes the genomics characterization of KPC-2-producing K. pneumoniae outbreak isolates from Shandong Province, China. In our study, these isolates appeared to be cloned, and ST11 K. pneumoniae was the major clone caused the outbreak. Therefore, routine surveillance of such strains in this region is urgently warranted.

Escherichia coli from Commercial Broiler and Backyard Chickens Share Sequence Types, Antimicrobial Resistance Profiles, and Resistance Genes with Human Extraintestinal Pathogenic Escherichia coli.

Escherichia coli recovered from poultry, and extraintestinal pathogenic E. coli (ExPEC), responsible for most cases of urinary tract infection (UTI) and bloodstream infection (BSI) in humans, may share genetic characteristics, suggesting that poultry are a potential source of ExPEC. Here, we compared E. coli isolated from commercial broiler and backyard chickens (n = 111) with ExPEC isolated from patients with community- or hospital-acquired UTI or BSI (n = 149) from Southeast Brazil. Isolates were genotyped by multilocus sequence typing, tested for susceptibility to antimicrobial agents, and screened for β-lactamase genes. We found that 10 genotypes were shared among poultry and human isolates: sequence type (ST) 10, ST48, ST58, ST88, ST90, ST93, ST131, ST602, ST617, and ST1018. Thirty-five (23%) ExPEC and 35 (31%) poultry E. coli isolates belonged to the shared STs. ST58 and ST88 isolates from human and poultry sources shared identical antimicrobial resistance profiles. blaTEM-1 was the most prevalent β-lactamase gene, identified in 65 (92%) of 71 ExPEC and 29 (67%) of 43 poultry E. coli that tested positive for β-lactamase genes. Commercial broiler chicken isolates shared the extended-spectrum β-lactamase (ESBL) genes blaCTX-M-2,blaCTX-M-8, and blaSHV-2 with human isolates; backyard chicken isolates lacked ESBL genes. In conclusion, several genotypic and phenotypic characteristics were shared between human and poultry E. coli; this suggests that there is potential for transmission of E. coli and antimicrobial resistance genes from poultry to humans, perhaps through environmental contamination, direct contact, or consumption. Additional research is needed to understand the potential direction and pathways of transmission.

High prevalence of carbapenemase, AmpC β-lactamase and aminoglycoside resistance genes in extended-spectrum β-lactamase-positive uropathogens from Northern India.

This study investigated the occurrence of extended-spectrum β-lactamase (ESBL) genes coexisting with carbapenemase, AmpC and aminoglycoside resistance gene in uropathogens in India. Antimicrobial susceptibility testing was performed by disk diffusion. Antimicrobial resistance genes were detected by multiplex PCR. Of 1516 consecutive urine samples, 454 (29.9%) showed significant bacteriuria with a single micro-organism, predominantly Escherichia coli (n=343), followed by Klebsiella pneumoniae (n=92), Pseudomonas aeruginosa (n=10) and Proteus mirabilis (n=9). Among the uropathogens, 61 ESBL-producers were identified containing blaCTX-M-15 (n=32), blaCTX-M-15+blaOXA-2 (n=15), blaCTX-M-15+blaOXA-2+blaTEM-1 (n=6), blaOXA-2 (n=5), blaOXA-2+blaSHV-76 (n=1), blaTEM-1+blaSHV-76 (n=1) and blaTEM-1 (n=1). All ESBL genes were located on horizontally transferable plasmids of incompatibility types HI1, I1, FIA+FIB, FIA and Y. Among the 61 ESBL-producers, 59 harboured carbapenemase genes, including blaNDM-5 (n=48), blaNDM-5+blaOXA-48 (n=5), blaNDM-5+blaIMP (n=5) and blaNDM-5+blaIMP+blaVIM (n=1). ESBL-producing uropathogens also harboured 16S rRNA methylase genes, including rmtB (n=9), rmtA (n=4), rmtC (n=1) and armA (n=1). ESBL-positive isolates also contained AmpC genes, including blaCIT (n=8) and blaDHA-1 (n=1). Imipenem and gentamicin had the lowest resistance rates against the uropathogens. This is the first report showing the high prevalence of carbapenemases in ESBL-positive isolates in this area. Regular surveillance for such resistance mechanisms will be useful for health personnel to treat infections by these multidrug-resistant pathogens.

Antimicrobial Resistance and Molecular Characterization of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Ducks in South Korea.

Ducks are potential carriers of pathogenic bacteria, which are capable of transmitting zoonotic diseases to humans. The global spread of Enterobacteriaceae carrying extended-spectrum β-lactamase (ESBL) genes is a public health concern. This study investigated the prevalence of antimicrobial resistance in Escherichia coli isolated from ducks in Korea and described the molecular characteristics of the ESBLs they produced. A total of 146 E. coli isolates from 404 duck fecal and carcass samples in 85 duck farms were tested for antimicrobial resistance using the broth dilution method and were further characterized using molecular methods. We observed high resistance rates to tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid, ampicillin, and ciprofloxacin. In total, six ceftiofur-resistant isolates (4.1%) were observed, which produced CTX-M-55 (n = 3) or CTX-M-65 β-lactamase (n = 3). All CTX-M-producing E. coli isolates were also resistant to ciprofloxacin, with mutations in the quinolone resistance determining region of GyrA (S83L with or without D87N) and ParC (S80I), and three CTX-M-producing E. coli isolates carried plasmid-mediated quinolone resistance (PMQR) genes, qepA (n = 1), qnrS, and acc(6')-Ib-cr (n = 2). The transfer of blaCTX-M genes was observed in one isolate mediated by IncF-family plasmids but not in the co-resistant isolates carrying both blaCTX-M and PMQR genes. Pulsed-field gel electrophoresis and multilocus sequence typing demonstrated that CTX-M-producing isolates were heterogeneous; however, identical isolates were found in different farms and slaughterhouses. This study presents baseline data on antimicrobial resistance of E. coli derived from duck samples and is the first report of CTX-M-55 and CTX-M-65 β-lactamase-producing E. coli isolated from ducks in Korea. The dissemination of ESBL-producing E. coli poses a potential risk to public health and therefore should be monitored.

Dynamics and molecular features of OXA-48-like-producing Klebsiella pneumoniae lineages in a Tunisian hospital.

The aim of this study was to elucidate the molecular features of genes, plasmids and clones of OXA-48-like producingKlebsiella pneumoniae isolates recovered in Sahloul Hospital (Sousse, Tunisia) in the period 2012-2014. In vitro antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and PCR-based replicon typing (PBRT) were performed. Extended-spectrum β-lactamase (ESBL) and carbapenemases genes were detected by PCR and sequencing. The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). Klebsiella pneumoniae accounted for 26.8% (1095/4083) of clinical Enterobacterales isolates identified during 2012-2014, of which 21.9% (240/1095) were resistant to carbapenems, mostly harbouring blaOXA-48-like genes (196/240; 81.7%). Plasmid analysis showed that blaOXA-204 and blaOXA-48 were mostly carried by IncA/C and IncL plasmids, respectively. The current data highlight the dominance of two ST101 and ST147 lineages spreading OXA-48 and OXA-204, respectively, through successive clonal spreads at this hospital. In addition, a large diversity of other K. pneumoniae lineages was also identified, such as ST15, ST36 and ST525 spreading OXA-48 as well as ST340, ST2032, ST301, ST199 and ST1561 spreading OXA-48 or OXA-204, constituting a reservoir of possible dominant clones in the future. This study reports the full molecular characterisation of carbapenem resistance in K. pneumoniae and the predominance of a few clones responsible for the dissemination of OXA-48 and OXA-204 enzymes in a Tunisian hospital.

Antimicrobial Susceptibility and Molecular Epidemiology of Proteus mirabilis Isolates from Three Hospitals in Northern Taiwan.

Of all the Proteus spp., Proteus mirabilis is the most common species identified in clinical specimens and is a leading agent of complicated urinary tract infection. This study was undertaken to understand the antimicrobial susceptibility, prevalence of antibiotic resistance genes, and molecular typing of P. mirabilis isolates collected from three hospitals in northern Taiwan. The results showed that the collected isolates of P. mirabilis were susceptible to most antibiotics except cefazolin and tigecycline. Many resistance genes were detected in the collected isolates, of which TEM genes were the most common. Resistance to third- or fourth-generation cephalosporins was related to the presence of at least one of the tested extended-spectrum β-lactamase (ESBL) or AmpC genes. The presence of the VEB-1 gene seemed to be a good predictor for both cefepime and ceftazidime resistance, which was further supported by quantitative polymerase chain reaction results. Of the four imipenem-resistant P. mirabilis isolates, three isolates could hydrolyze imipenem by mass spectrometry analysis. Molecular typing by pulsed-field gel electrophoresis showed that the pulsotyping of the selected P. mirabilis isolates was heterogeneous. By analyzing the relationship of antimicrobial resistance and the presence of resistance genes, revision of the Clinical and Laboratory Standards Institute cefepime and ceftazidime MIC breakpoints for Enterobacteriaceae to predict ESBL producers might possibly be needed.

Comparative Activities of Ceftazidime-Avibactam and Ceftolozane-Tazobactam against Enterobacteriaceae Isolates Producing Extended-Spectrum β-Lactamases from U.S. Hospitals.

The activities of ceftazidime-avibactam, ceftolozane-tazobactam, and comparators were evaluated for 733 isolates displaying resistance to broad-spectrum cephalosporins and carrying extended-spectrum β-lactamase (ESBL) genes detected by whole-genome sequencing analysis. Isolates were collected during 2017 in U.S. hospitals. The ESBL producers were 486 Escherichia coli, 190 Klebsiella pneumoniae, and 42 Enterobacter cloacae isolates and isolates from 3 other species. The most common groups of ESBL-encoding genes were blaCTX-M-15-like (n = 491 isolates) and blaCTX-M-15 alone (n = 168) or plus blaOXA-1 (n = 260), followed by blaCTX-M-14-like (n = 162), which included blaCTX-M-27 and blaCTX-M-14 (104 and 51 isolates, respectively), and blaSHV-12 and blaSHV-7 (48 and 22 isolates, respectively). ESBL producers carried other β-lactamases, including 1 E. cloacae harboring blaKPC-3 All ESBL-producing isolates were susceptible to ceftazidime-avibactam, and 90.2/83.9% (CLSI/EUCAST breakpoints) were susceptible to ceftolozane-tazobactam. Tigecycline (98.1/95.8% susceptible) and colistin (99.2%) were comparators that displayed the greatest activity against these isolates. Ceftolozane-tazobactam inhibited 91.4/83.9% of isolates carrying blaCTX-M-15-like and 97.5/95.1% of isolates carrying blaCTX-M-14-like, and its activity was more limited against the 91 isolates carrying blaSHV (66.7/61.1% susceptible). Ceftolozane-tazobactam inhibited 95.5% of the E. coli isolates but only 83.0%, 64.3%, and 80.0% of K. pneumoniae, E. cloacae, and other species harboring ESBL-encoding genes (CLSI breakpoints), respectively. Outer membrane protein sequences for ceftolozane-tazobactam-nonsusceptible isolates did not exhibit significant differences compared to those in genetically related ceftolozane-tazobactam-susceptible isolates. Ceftazidime-avibactam was more active than other agents tested, including ceftolozane-tazobactam, and the activity of this combination was stable regardless of species or ESBL gene carried.

Enterohaemorrhagic Escherichia coli O121:H19 acquired an extended-spectrum β-lactamase gene during the development of an outbreak in two nurseries.

Enterohaemorrhagic Escherichia coli (EHEC) is an important human pathogen worldwide. Although serotype O157 is currently the most dominant and important EHEC strain, serotypes O26, O111, O91, O103 and O121 are also recognized as serious pathogens that affect public health. EHEC outbreaks often occur in nurseries and elderly care facilities. In 2012, a nursery outbreak of EHEC O121 occurred during which the bacterium acquired a plasmid-borne extended-spectrum β-lactamase (ESBL) gene. ESBL-producing E. coli O86 was concurrently isolated from one of the EHEC patients. Therefore, we investigated the isolates by whole-genome sequence (WGS) analysis to elucidate the transmission dynamics of the EHEC strains and the ESBL plasmid. According to WGS-based phylogeny, all 17 EHEC O121 isolates were clonal, while E. coli O86 was genetically distant from the EHEC O121 isolates. The complete sequence of an ESBL plasmid encoding the CTX-M-55 β-lactamase was determined using S1-PFGE bands, and subsequent mapping of the WGS reads confirmed that the plasmid sequences from EHEC O121 and E. coli O86 were identical. Furthermore, conjugation experiments showed that the plasmid was capable of conjugative transfer. These results support the hypothesis that EHEC O121 acquired an ESBL-producing plasmid from E. coli O86 during the outbreak. This report demonstrates the importance of implementing preventive measures during EHEC outbreaks to control both secondary infection and the spread of antimicrobial resistance factors.

Characterization of Carbapenemase-Producing Klebsiella oxytoca in Spain, 2016-2017.

There is little information about carbapenemase-producing (CP) Klebsiella oxytoca, an important nosocomial pathogen. We characterized CP K. oxytoca isolates collected from different Spanish hospitals between January 2016 and October 2017. During the study period, 139 nonduplicate CP K. oxytoca isolates were identified; of these, 80 were studied in detail. Carbapenemase and extended-spectrum β-lactamase genes were identified by PCR and sequencing. Genetic relatedness was studied by pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS), carried out on 12 representative isolates, was used to identify the resistome, to elucidate the phylogeny, and to determine the plasmids harboring carbapenemase genes. Forty-eight (60%) isolates produced VIM-1, 30 (37.5%) produced OXA-48, 3 (3.7%) produced KPC-2, 2 (2.5%) produced KPC-3, and 1 (1.2%) produced NDM-1; 4 isolates coproduced two carbapenemases. By PFGE, 69 patterns were obtained from the 80 CP K. oxytoca isolates, and four well-defined clusters were detected: cluster 1 consisted of 11 OXA-48-producing isolates, and the other three clusters included VIM-1-producing isolates (5, 3, and 3 isolates, respectively). In the 12 sequenced isolates, the average number of acquired resistance genes was significantly higher in VIM-1-producing isolates (10.8) than in OXA-48-producing isolates (2.3). All 12 isolates had chromosomally encoded genes of the blaOXY-2 genotype, and by multilocus sequence typing, most belonged to sequence type 2 (ST2). Carbapenemase genes were carried by IncL, IncHI2, IncFII, IncN, IncC, and IncP6 plasmid types. The emergence of CP K. oxytoca was principally due to the spread of VIM-1- and OXA-48-producing isolates in which VIM-1- and OXA-48 were carried by IncL, IncHI2, IncFII, and IncN plasmids. ST2 and the genotype blaOXY-2 predominated among the 12 sequenced isolates.